Under the tenure of grant RO1-AI062894 our laboratory developed a new concept and vaccine approach to induce protective CD8 T cell responses against viruses using influenza A virus as model system. The new cell-based vaccine is a unique form of immunization best described as transgenic antigen-presenting cells (APCs) vaccine. Using this system we established that central memory CD8 T cells mediate protection against disease caused by influenza virus. In this revised competing renewal application we will capitalize on the body of information generated in past years and ask new questions designed to better understand the requirements for the induction of central memory CD8 T cells in vivo by vaccination. Three aims are directed at this question. In AIM 1, Direct vs. cross-priming memory CD8 T cell induction by transgenic APCs, using different vaccination modalities we will investigate and distinguish the role of direct presentation, cross-priming by an [unreadable]APC feeding the APC[unreadable] cross-priming mechanism, and crosspriming by antigen synthesized and secreted by the APC, on the generation of central memory CD8 T cells. In Aim 2, Reactivation of TCM cells: Correlation with the modality of vaccination, we will analyze the expansion of already established memory T cells to understand which mechanisms is best in the reactivation of resting memory T cells. In Aim 3, MicroRNAs manipulation to facilitate central memory T cell fate determination, we will investigate a new approach to program from the outset central memory CD8 T cell fate: microRNAs modulation. This revised competing renewal application has been refocused on two main areas with one main common goal: test vaccines and vaccination strategies to selectively generate and expand anti-virus central memory CD8 T cells. It is expected that these studies will broaden our understanding on the principles for optimal generation of protective anti-virus CD8 T cell responses by cell-based vaccination. The experimenst may lead to the development of new safe vaccines against influenza A virus and viruses in general.